One of the major problems hindering development of gene therapy approaches, including the use of RNAi for gene silencing, is the relative lack of methods to deliver genes/RNAi to specific cell types. This application addresses this limitation. Our major interest is to develop gene therapy approaches to treat acute lung injury, which accounts for over 50,000 deaths each year in the US. We have developed a highly effective treatment for this disease in mouse and pig models that uses the ubiquitous overexperssion of the Na+,K+-ATPase to increase alveolar fluid clearance from the previously injured lung. Our experiments show that this treatment not only improves edema resolution (and lung function and survival), but also improves alveolar epithelial/endothelial barrier function by upregulating tight junction complexes. However, we do not know in what cells transfer and expression of the Na+,K+-ATPase gene is needed for this protection and treatment, nor whether upregulation of tight junction complexes is needed for maximal effect. In order to understand the mechanisms responsible for this treatment approach, we must be able to deliver genes specifically to the individual cell types in the alveolus and silence gene expression in a similar cell-specific manner. Unfortunately, there are no uniformly effective ways to deliver RNAi to specific cell types in vivo. We have developed a way to overcome this problem. We have shown that the nuclear localization of plasmids in the absence of cell division is sequence-specific and requires transcription factors for nuclear import. We have identified a number of DNA sequences that show cell-specificity of nuclear import because they bind to cell- specific transcription factors. These include sequences that act in endothelial cells, alveolar type 1, and alveolar type 2 epithelial cells in vitro and in living animals. We have used these sequences to overexpress genes in these cells in vivo and will now use them to silence genes by shRNA delivery. This is an entirely new way to direct cell-specific RNAi delivery. Our aims are to (1) Determine in which alveolar cell types gene transfer of the Na+,K+-ATPase is needed for improved alveolar fluid clearance and induction of tight junctions, (2) Test whether tight junctions are needed for Na+,K+-ATPase gene transfer-mediated protection and treatment of acute lung injury by developing a method for cell-specific RNAi delivery, and (3) Determine whether gene transfer of tight junction complex proteins alone can treat lung injury or further enhance Na+,K+- ATPase gene transfer-mediated treatment of acute lung injury.